Comparison Of Hematopoiesis-supportive Function Of Human Umbilical Cord Blood-derived Stromal Cells And Mesenchymal Stem Cells

IntroductionNow, human umbilical cord blood as an alternative and attractive source is often usedfor cellular or gene therapy, all these due to its abundant, collected easily, low probability ofpathophoresis, low malignant cells contaminate and low immunogenicity.Human umbilical cord blood derived stromal cells (hUCBDSCs) that we havesuccessfully isolated and cultured, is mainly composed of macrophage-like, fibroblastlike,and small-sphere-like cells. In our prior research on support hematopoietic, the data showsthat hUCBDSCs can secrete thrombopoietin (TPO), stem cell factor (SCF) and granulocytemacrophage colony-stimulating factor (GM-CSF) to support the development ofhematopoietic stem cells. Particularly, hUCBDSCs is superior to human bone marrowstromal cells (hBMSCs) in secreting TPO and the expansion of colony-forming unit ofmegakaryocyte (CFU-Mk) in vitro and in vivo.Mesenchymal stem cells, as the precursor cells of stromal cell, have beendemonstrated existing in human umbilical cord blood by several research groups. Humanumbilical cord blood derived mesenchymal stem cells (hUCBDMSCs) obtained fromcolony forming unit fibroblasts (CFU-F) have homogeneous morphology, express some CDmolecule consisting with bone marrow derived mesenchymal stem cells, possess thepotential to support vitro of hematopoietic stem/progenitor cells proliferation anddifferentiation, and the capacity of differentiating into classical three connective tissuelineages. Additional, hUCBDMSCs also secreted some hematopoietic cytokines.Stromal cells and mesenchymal stem cells are two improtant cell populations withinhematopoietic microenviroment, play an important role in the development ofhematopoietic stem/progenitor cell. However the biological characteristics betweenhUCBDSCs and hUCBDMSCs have not yet been identified, particularly, the application ofhUCBDSCs and hUCBDMSCs on supporting hematopoiesis is still discussed. In order to prompt our recognization and reasonable using the two cell populations, so in this research,we focused on some biological characteristics of hUCBDSCs and the capacity of supporthematopoiesis in ex vivo in comparison with hUCBDMSCs.MethodsPart one: hUCBDSCs/hUCBDMSCs biological characteristicsMononuclear cell separation of hUCBThe mononuclear cells were separated from full-term infants umbilical cord blood.Cell separation was performed according to the follow, the majority of red blood cell wasdepleted by6%gelatin sedimentation method, the leukocyte-rich fraction was centrifuged,the deposition was loaded on the same volum of Percoll density gradient fractionationcolumns scrupulously (density=1.077g/l). Cells were centrifuged at2000×rpm for20minutes at4°C. The mononuclear cells (MNCs) at the interface were centrifuged andwashed by PBS. The quantity of the hUCB MNCs from0.5×10~7to12.5×10~7. All thesemanipulation were finished within4hours.Establishment of hUCB-derived stromal cellsThe hUCB MNCs were cultured in modified Dexter system to obtain hUCB-derivedstromal cells(hUCBDSCs). MNCs were resuspended in DMEM/F12medium containing12.5%fetal bovine serum,12.5%horse serum,10μM hydrocortisone, supplemented withstreptomycin (100ng/ml) and penicillin (100U/ml). Culture was replaced by fresh mediumafter48hours, adherent cells were left, and then, half medium was changed by freshmedium weekly. After the density of adherent cells had risen above80%confluence,hUCBDSCs were subcultured with1:2ratio under the same culture medium and condition.Establishment of hUCB-derived mesenchymal stem cellsThe hUCB MNCs were cultured in mesenchymal stem cell medium(MSCM) to obtainhUCB-derived mesenchymal stem cells (hUCBDMSCs). MNCs were resuspended inMSCM, culture was replaced by fresh medium after48h, then adherent cells weredemi-depopulated weekly and fresh medium was added. After the density of adherent cellshad risen above80%confluence, hUCBDMSCs were subcultured with1:3ratio under thesame culture medium and condition.Colony-forming unit-fibroblast (CFU-F) assay1×10~7mononuclear cells, isolated from UCB were seeded in modified Dexter system medium or MSCM in T-25flasks, and incubated in a humidified atmosphere with5%CO2at37°C. The medium was demi-depopulated every week. The fibroblast colonies werecounted on day10of culture with an inverted microscope. Cell clusters containing>50cells were scored as CFU-F colonies.Detecting cell cycle of hUCB-derived stromal cells and hUCB-derivedmesenchymal stem cells by flow cytometerCells were collected after digestion with0.25%trypsogen and prepared in normalmethods, then, analyzed by FACS flow cytometer. The cell cycle distribution (G0/G1, S andG2~+M) was quantified by the software Cell Fit2.0and2.1.Cells proliferation assayCells proliferation assays were performed using the Cell Counting Kit-8.100μl ofsingle-cell suspension from passage two,5×104/ml, was added into a96-well plate and withtriplicate wells in each group, and medium were used as controls. The cells were cultured at37°C with5%CO2for24h. At the following indicated time points, the cell numbers intriplicate wells were measured as the absorbance (450nm).Phenotypic AnalysisCells from hUCBDSCs and hUCBDMSCs respectively were stained withphycoerythrin (PE)-conjugated antibodies against CD106, CD34or fluoresceinisothiocyanate (FITC)-conjugated antibodies against Fn, Lm, CD45, CD44, CD29, Stro-1.Relative mouse isotypic antibodies served as the control. Cells were stained in single labeland then analyzed by flow cytometry with a FAC Scan.Multi-lineage differentiation assaysThe potency of hUCBDSCs and hUCBDMSCs differentiation into various cell typesincluding osteoblast, chondrocytes, and adipocytes were examined following StemproOstegenesis, Chondrogenesis, Adipogenesis Differentiation Kit recommended methods.After differentiation under proper stimuli, the multilineage potential was evaluated byAlizarin Red S staining for osteoblast, Alcian Blue staining for chondrocyte, and byaccumulation of lipid-rich vacuoles and Oil Red O staining for adipocytes on21day.Part two: the capacity of support hematopoiesis in ex vivoQuantitative real-time polymerase chain ractionTotal RNA from hUCBDSCs or hUCBDMSCs was extracted using Trizol reagent according to the manufacturer's instructions. The RNA precipitate was then dissolved in20μl of RNAse free water and analyzed for quantity and quality using a spectrophotometer.The integrity of the total RNA was examined by1%agarose gel electrophoresis, thequantity was determined based on absorbance at260nm (A260), and the purity wasanalyzed based on the absorbance ratio at260and280nm (A260/280). The cDNA wassynthesized from1μg of total RNA using PrimeScript RT reagent Kit. Five genes weretested by common PCR and real-time PCR including: granulocyte colony-stimulating factor(G-CSF), stem cell fator (SCF), thrombopoietin (TPO), stromal cell-derived factor1(SDF-1), granulocyte macrophage colony-stimulating factor (GM-CSF). Common PCR wasrun on PCR amplification apparatus, the PCR products were examined by1%agarose gelelectrophoresis, β-actin served as an internal control. Quantitative real-time PCR was runon an ABI7500real-time PCR system according to the manufacturer's recommendations.We used the SYBR Green for all the genes that we tested. GAPDH served as an internalcontrol. Data analysis was performed using the Sequence Detector System software (ABI)and the results were expressed as fold-change in relative mRNA expression level. Eachevaluation was performed in triplicate in the independent experiments.Enzyme linked immunosorbent assay (ELISA)ELISA was performed according to the manufacturer's instructions to analyze theconcentration of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophagecolony-stimulating factor (GM-CSF), stem cell fator (SCF), stromal cell-derived factor1(SDF-1), thrombopoietin (TPO) in the supernatants of hUCBDSCs and hUCBDMSCs wheneach group cells had risen above80%confluence respectively. After γ-irradiation at a doseof12Gy for each group cells, coculture with hematopoiesis cells, on0,7,14day, weanalyze the concentration of cytokins.Coculture of hematopoietic cells colony AssaysMononuclear cells were isolated from hUCB using Percoll density gradientfractionation. After culture, nonadherent CD34~+fraction in suspended MNCs wereharvested. After γ-irradiation at a dose of12Gy,2.0×105hUCBDSCs,2.0×105hUCBDMSCs were seeded in24-well plates respectively as different feeder layer cells.After24hours,1×106MNCs were plated respectively. At the5th day, nonadherentexpanded cells were inoculated in methylcellulose-based semisolid cultures. Cultures were maintained at37℃and5%CO2humidified air. After7-14days of culture, cells clonogenicpotential were assessed by erythroid colony-forming units (CFU-E), granulocyte/macropha-ge colony-forming units (CFU-GM) and CFU-GEMM using an inverted microscope, noncoculture CD34~+fraction in suspended MNCs were used as control. Colonies were countedand evaluated accordingly to manufacturer's instructions.Coculture of hematopoietic stem/progenitor cells phenotypic analysisUCB MNC were CD34~+-enriched using immuno-magnetic cell sorting. After analyzedthe percentage of CD34~+cells by flow cytometry, hematopoietic stem/progenitor cells wereseeded in T-25flasks which confluent hUCBDSCs or hUCBDMSCs growth-inactivated, at37℃and5%CO2humidified air for14days.Hematopoietic stem/progenitor cells were analyzed by flow cytometry on7th and14th coculture day, using a panel of monoclonal antibodies (FITC-, or PE-conjugated):CD34for stem/progenitor cells; CD14, CD15and CD33for myeloid lineage, CD3, CD19for lymphocytes and CD41a for megakaryocytes, CD71for erythrocytes. Isotype controlswere also prepared for every experiment.Result:Part one: hUCBDSCs/hUCBDMSCs biological characteristicsDynamic observation of hUCB-derived stromal cells and hUCB-derivedmesenchymal stem cells in cultureThe hUCB MNCs were cultured in modified Dexter system. By3–4days, themorphology of the cultured cells varied from round, triangular, fusiform, or irregular. Withthe period of culture prolonged, cells were mainly composed of macrophage-like,fibroblast-like, and small-sphere-like cells. Stromal cells did not form colonies duringculture period. hUCBDSCs can be subcultured up to3generations under the sameconditions. In the limited passages, cell morphology remained heterogeneous. With thenumber of passages increase, the confluence delayed.When the hUCB MNCs were cultured in MSCM, at the first week, the shapes ofadherent cells appeared as small fusiform, triangular, or spherical cells. When culture about10days, a few fibres spindle cells developed dense colony and began rapid growth,expansion, whereas macrophage-like cells, others spindle cells and small-sphere-like cellsdecrease greatly, cell morphology tended to be homogeneouse. By28day, hUCBDMSCs increased significantly and filled the culture flask, The hUCBDMSCs could be readilyexpanded in vitro by serial passage every4-10days for12passages, without visiblechanges in either the growth patterns or morphology. With the number of passages increase,the confluence delayed gradually.12of18hUCB MNCs formed fibroblast-like colonies inMSCM, the mean number of adherent fibroblastlike colonies was0.42±0.07/106monuclearcells.Cell cycle and growth curveThe results of hUCBDSCs cycle indicated that76.18%cells were in G0/G1phase,9.87%cells were in S phase, and13.94%cells were in G2~+M phase. HUCBDMSCs cycledemonstrated that86.34%cells were in G0/G1phase,4.14%cells were in S phase, and9.61%cells were in G2~+M phase.The growth curve showed that hUCBDSCs were in dormancy and adaptation phasein the first4days in culture. Then, the cells came into a logarithm growth phase andreached their peak at the8th day. After that, the cells decreased a bit and their growthentered a stable period. HUCBDMSCs were in dormancy and adaptation phase in the first2days in culture. Then, the cells came into a logarithm growth phase and reached their peakat the4th day. After that, the cells decreased a bit and their growth entered a stable period.Immunophenotype of hUCB-derived stromal cells and mesenchymal stem cellshUCBDSCs shared some of their immunophenotype with hUCBDMSCs, includingpositivity for Fn, CD44and Stro-1, negativity for CD34. However, Lm and CD29expressedin hUCBDMSCs, not in hUCBDSCs. CD45and CD106expressed in hUCBDSCs, not inhUCBDMSCs.Multi-lineage differentiation capacitiesDifferentiation of the hUCBDSCs and the hUCBDMSCs cells were assessed usingfrom passage2to passage5. Cells kept in the regular growth medium served as the control.Osteogenic,adipogenic and chondrogenic differentiation were detected in HUCBDMSCsgroup, HUCBDMSCs did not induce to the tri-lineage cells.Part two: the capacity of support hematopoiesis in ex vivoGene expression of growth factors by hUCB-derived stromal cells andhUCB-derived mesenchymal stem cellshUCBDSCs and hUCBDMSCs expressed G-CSF, TPO, SCF, SDF-1and GM-CSF gene, hUCBDSCs expressed lower levels in TPO, SCF, SDF-1and GM-CSF, but expressedhigher level in G-CSF gene compared with hUCBDMSCs.Cytokine secretion of hUCB-derived stromal cells and hUCB-derivedmesenchymal stem cellshUCBDSCs and hUCBDMSCs dynamically secreted G-CSF, TPO GM-CSF, SCF andSDF-1when coculture with hematopoiesis cells. On day0, the level of G-CSF was higherin hUCBDSCs group compared with hUCBDMSCs group; the level of SCF was lower inhUCBDSCs group compared with hUCBDMSCs group; the level of TPO, GM-CSF andSDF-1was comparable. The level of G-CSF, SCF and SDF-1decreased on day7, thenincreased on the14th day in each groups. The level of TPO in each groups showed increasemarkedly during14days, hUCBDSCs group was higher than hUCBDMSCs group at thelast two time pionts. The level of GM-CSF in hUCBDMSCs group showed decrease on day7, increase on day14, while it increased gradually in hUCBDSCs group. In addition, thelevel of GM-CSF in hUCBDSCs group was significant higher than that in hUCBDMSCsgroup at the last two time points.Expansion of hUCB-derived hematopoietic cells on hUCB-derived stromal cellsfeeder layer and hUCB-derived mesenchymal stem cells feeder layerAfter co-cultured hematopoietic cells5days, colony counts of CFU-GM ofhematopoietic cells from hUCBDSCs feeder layer were more than that from hUCBDMSCs;CFU-GEMM counts from hUCBDMSCs feeder layer were more than that from hUCBDSCsfeeder layer; the control group was deficiency in forming colonies. hUCBDSCs wassuperior to hUCBDMSCs in enhancing expansion of CFU-GM (P<0.01).Hematopoietic stem cells committed differentiationThe averaged percentage of sorting CD34~+hematopoietic stem cells (HSCs) in theCD34~+-enriched cells was90.2%by flow cytometry analyzing.On day7, the percentage of CD34~+cells was decreased in different groups. Theexpression of CD19, CD3and CD41a were negative, while CD33, CD71and CD14werepositive. The expression of CD15was positive in hUCBDSCs group, negative inhUCBDMSCs group (p=0.008). The level of CD33and CD71in hUCBDMSCs werecomparable with that in hUCBDSCs (p=0.539and p=0.813respectively). On day14, thepercentage of CD34~+cells was further decreased. The expression of CD19and CD41a were still negative, CD15, CD3, CD33, CD71and CD14were positive. The level of CD33andCD71respectively increased and decreased significantly. CD15increased in hUCBDSCsgroup, but the level of CD15was lower than that in hUCBDMSCs group (p=0.0005). ThehUCBDSCs group was superior to hUCBDMSCs group in inducing CD34~+HSCsdifferentiated into CD33~+myelocyte and CD3~+lymphocyte (p=0.001and p=0.004respectively). In this phase, the expression of erythrocyte CD71decreased significantly.Conclusion1. The optimal medium is an important factor to successful culture hUCBDSCs/hUCBDMSCs. hUCBDSCs are different from hUCBDMSCs in cell biological characters,hUCBDMSCs have more stem cell characters than hUCBDSCs. hUCBDMSCs have thecapacity of classic tri-lineage differentiation in induce condition.2. hUCBDSCs possessed obtaining easily, secreted higher level of G-CSF, TPO andGM-CSF, inducing hematopoiesis stem cells differentiation into myeloid lineage, especiallygranulocyte lineage, at earlier coculture stage in vitro. Furthermore, hUCBDSCs possessedstrengthen potency in promoting thrombocytopoiesis. In one word, hUCBDSCs are superiorto hUCBDMSCs in inducing myeloid lineage differentiation in vitro.